4.2. Production of Tryptic Hydrolysate of Whey Proteins

The tryptic hydrolysate was prepared according to the protocol of Pouliot et al. [25]. Briefly, trypsin solution (Trypsin VI) from Neova technologies (Abbotsford, BC, Canada) was solubilized at 10% (w/v) in HCl (0.001 N) and it was added to whey protein solutions at an enzyme-to-substrate (E/S) ratio of 1/700. Hydrolysis was carried out at 38 °C, pH 8 using the pH-stat method. When a degree of hydrolysis (DH) of 5.6% was reached, the enzymatic reaction was stopped by separating the reaction products (tryptic peptides) from the enzyme and substrate (non-hydrolyzed proteins) by UF (polyethersulfone membrane with MWCO of 10 kDa) while using a GEA Niro system from GEA Niro Inc. (Düsseldorf, Germany). UF was performed for 100 min at 35.5 °C under a constant transmembrane pressure (TMP) of 4.5 bar. During UF, a discontinuous diafiltration (DF) was carried out. The UFr was diluted with 1 diavolume (DV, Equation (1)) twice and the diluted UFr was concentrated to a final volume concentration factor (VCF) of 10×.

where Vw corresponds to the volume (L) of water added to retentate and VUFr is the volume (L) of UFr.

After UF, the three batches of tryptic hydrolysate of whey proteins (UFp) were stored at 4 °C until concentration by RO. The samples of hydrolysates produced before UF (H), as well as after UFp and UFr from each batch, were also collected, freeze-dried, and stored at −18 °C until analysis.